exemestane’s17-hydroxylatedmetaboliteexertsbiological ... › content › molcanther › ...nov...

Exemestane’s 17-hydroxylated metabolite exerts biologicaleffects as an androgen

Eric A. Ariazi,1,2 Andrei Leitão,3 Tudor I. Oprea,3

Bin Chen,1 Teresa Louis,1 Anne Marie Bertucci,1

Catherine G.N. Sharma,2 Shaun D. Gill,2

Helen R. Kim,2 Heather A. Shupp,2 Jennifer R. Pyle,2

Alexis Madrack,2 Anne L. Donato,2 Dong Cheng,1

James R. Paige,1 and V. Craig Jordan1,2

1Robert H. Lurie Comprehensive Cancer Center, NorthwesternUniversity Feinberg School of Medicine, Chicago, Illinois;2Fox Chase Cancer Center, Philadelphia, Pennsylvania; and3Division of Biocomputing, University of New Mexico HealthSciences Center, Albuquerque, New Mexico

AbstractAromatase inhibitors (AI) are being evaluated as long-term adjuvant therapies and chemopreventives in breastcancer. However, there are concerns about bone mineraldensity loss in an estrogen-free environment. Unlikenonsteroidal AIs, the steroidal AI exemestane mayexert beneficial effects on bone through its primarymetabolite 17-hydroexemestane. We investigated 17-hydroexemestane and observed it bound estrogen recep-tor A (ERA) very weakly and androgen receptor (AR)strongly. Next, we evaluated 17-hydroexemestane inMCF-7 and T47D breast cancer cells and attributeddependency of its effects on ER or AR using theantiestrogen fulvestrant or the antiandrogen bicalutamide.17-Hydroexemestane induced proliferation, stimulatedcell cycle progression and regulated transcription at highsub-micromolar and micromolar concentrations throughER in both cell lines, but through AR at low nanomolarconcentrations selectively in T47D cells. Responses of

each cell type to high and low concentrations of the non-aromatizable synthetic androgen R1881 paralleled thoseof 17-hydroexemestane. 17-Hydroexemestane down-regulated ERA protein levels at high concentrations in acell type–specific manner similarly as 17B-estradiol, andincreased AR protein accumulation at low concentrationsin both cell types similarly as R1881. Computer dockingindicated that the 17B-OH group of 17-hydroexemestanerelative to the 17-keto group of exemestane contributedsignificantly more intermolecular interaction energy to-ward binding AR than ERA. Molecular modeling alsoindicated that 17-hydroexemestane interacted with ERAand AR through selective recognition motifs employed by17B-estradiol and R1881, respectively. We conclude that17-hydroexemestane exerts biological effects as anandrogen. These results may have important implicationsfor long-term maintenance of patients with AIs. [MolCancer Ther 2007;6(11):OF1–11]

IntroductionThe third-generation aromatase inhibitors (AI) anastrozole(Arimidex; refs. 1, 2), letrozole (Femara; refs. 3, 4), andexemestane (Aromasin; refs. 5, 6), by virtue of blockingextragonadal conversion of androgens to estrogens andgiving rise to an estrogen-depleted environment, exhibitimproved efficacy over tamoxifen in the adjuvant therapyof estrogen receptor (ER)–positive breast cancer in post-menopausal women (7). Clinical trials evaluating these AIsshowed a reduced incidence of contralateral primary breastcancer in the AI groups compared with tamoxifen (1–6);hence, AIs are currently being evaluated as chemopreven-tives in ongoing studies (8). AIs also exhibit reduced overalltoxicity compared with tamoxifen (1–6, 9), but the toxicityprofiles are different: tamoxifen is associated with in-creased incidences of thromboembolic events and endome-trial cancer, whereas AIs are associated with decreasedbone mineral density (BMD), coupled with an increasedrisk of bone fractures (10–12) and severe musculoskeletalpain that limits patient compliance (13, 14). Because theavailable third-generation AIs all exhibit similar efficacies,the selection of a specific AI for long-term adjuvant therapyof breast cancer and as a chemopreventive in healthywomen at high risk for breast cancer will likely bedetermined by safety and tolerability profiles.AIs fall into two classes, steroidal as represented by

exemestane, which acts as a suicide inhibitor of aromatase,and nonsteroidal including anastrozole and letrozole,which reversibly block aromatase activity (7). Possiblydue to its steroid structure, exemestane may exhibit aunique pharmacology distinct from the nonsteroidal AIs.In two preclinical studies by Goss et al. (15, 16), exemestanewas given to female ovariectomized rats, an animal model

Received 5/3/07; revised 8/28/07; accepted 10/1/07.

Grant support: Department of Defense Breast Program under awardBC050277 Center of Excellence (V.C. Jordan; views and opinions of,and endorsements by the author(s) do not reflect those of the U.S. Armyor the Department of Defense), Specialized Programs of ResearchExcellence in Breast Cancer CA89018 (V.C. Jordan), the Avon Foundation(V.C. Jordan), the Weg Fund (V.C. Jordan), and NIH P30 CA006927(Fox Chase Cancer Center), an Eli Lilly Fellowship (Robert H. LurieComprehensive Cancer Center), the Lynn Sage Breast Cancer ResearchFoundation (Robert H. Lurie Comprehensive Cancer Center), the NIHMolecular Libraries Initiative award U54 MH074425-01, and by NationalCancer Institute CA118100 (University of New Mexico Cancer Center).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Requests for reprints: V. Craig Jordan, Alfred G. Knudson Chair of CancerResearch, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia,PA 19111-2497. Phone: 215-728-7410; Fax: 215-728-7034.E-mail: [email protected]

Copyright C 2007 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-07-0312

OF1

Mol Cancer Ther 2007;6(11). November 2007

Published Online First on November 7, 2007 as 10.1158/1535-7163.MCT-07-0312

on June 10, 2021. © 2007 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst November 7, 2007; DOI: 10.1158/1535-7163.MCT-07-0312

http://mct.aacrjournals.org/

of osteoporosis, and found to reduce bone resorptionmarkers and increase BMD and bone strength, whereaslowering serum cholesterol and low-density lipoproteinlevels compared with ovariectomized controls. One of thesepreclinical studies also evaluated the nonsteroidal AIletrozole, but in contrast, found no benefit of letrozole onbone or lipid profiles (16). In a clinical study investigatingthe effects of 2 years of exemestane on bone compared withplacebo without prior tamoxifen therapy in patients withsurgically resected breast cancer at low risk for recurrence,exemestane did not enhance BMD loss in lumbar spine andonly modestly enhanced BMD loss in the femoral neckcompared with the placebo group (17). Interestingly, in thisstudy, exemestane promoted bone metabolism by increas-ing levels of both bone resorption and formation markers(17). However, a clear-cut advantage of exemestane versusthe nonsteroidal AIs on bone safety has not been shown inhumans, possibly because all other clinical studies com-pared the AI to tamoxifen (9, 12, 18) or the AI to placebowith prior tamoxifen therapy (10, 11). Drawing conclusionsfrom these studies is difficult because tamoxifen preservesBMD, thereby protecting against fractures, and withdrawalof tamoxifen may have lasting effects on BMD (19).Maintenance of BMD in women is a known estrogenic

effect (20). However, androgen receptors (AR) are alsoexpressed in multiple bone cell types (21, 22), and studiesshow that androgens maintain BMD in ovariectomizedrats (23, 24) and in women (21, 25–27). In ovariectomizedrats, physiologic concentrations of androstenedione, aweak androgen and a substrate of aromatase, reducedloss of bone, and the antiandrogen bicalutamide abrogatedthis effect (23), but anastrozole did not (23). Therefore, theprotective effect of androstenedione on maintenance ofBMD was androgen mediated and not due to aromatizationof androstenedione to estrogen. Furthermore, the non-aromatizable androgen 5a-dihydrotestosterone has beenshown to stimulate bone growth in osteopenic ovariecto-mized rats (24). In pre- and postmenopausal women,endogenous androgen levels correlate with BMD (25, 26).Furthermore, a study comparing estrogen to a syntheticandrogen in postmenopausal osteoporotic women showedthat both steroids were equally effective in reducing boneresorption (27). Also, a 2-year double-blind trial showedthat estrogen plus a non-aromatizable androgen signifi-cantly improved BMD over estrogen alone in surgicallymenopausal women (28). Therefore, exogenous androgenspromote BMD maintenance in women when used alone(27) and in conjunction with estrogen (28).Although exemestane does not bind ER, it is structurally

related to androstenedione and has weak affinity for AR(29, 30). At high doses, exemestane exerts possibleandrogenic activity in vivo by inducing an increase inventral prostate weight in immature castrated rats (29).Recently, Miki et al. (22) showed in human osteoblast hFOBand osteosarcoma Saos-2 cells that exemestane promotedproliferation, which was partially blocked by the anti-androgen hydroxyflutamide, and increased alkaline phos-phatase activity. However, metabolites of exemestane may

be mediating these effects. Exemestane is given p.o. at25 mg/day and rapidly absorbed, showing peak plasmalevels within 2 to 4 h and a direct relationship betweendosage and peak plasma levels after single (10–200 mg) orrepeated doses (0.5–50 mg; refs. 30, 31). Single-dose studiessuggested that exemestane has a short elimination half-life,but multiple-dose studies show its terminal half-life to beabout 24 h. Exemestane undergoes complex metabolism,and the primary metabolite in plasma has been identifiedas 17-hydroexemestane, which accumulates to a concen-tration of about 10% of its parent compound (30). Takingthe possible action of metabolites into consideration, Gosset al. (16) administered 17-hydroexemestane to ovariecto-mized rats and found that it produced the same bone-sparing effects and favorable changes in circulating lipidlevels as exemestane. Also, Miki et al. (22) stated that17-hydroexemestane promoted proliferation of the osteo-blast and osteosarcoma cells similar to exemestane, but thedata were not shown, and the authors did not furtherexplore 17-hydroexemestane activities. Additionally, Mikiet al. (22) showed that the osteoblasts efficiently metabo-lized androstenedione to testosterone, which involves thereduction of the 17-keto group of androstenedione to ahydroxyl group. Similar metabolism would convertexemestane to 17-hydroexemestane, and thus, activities ofexemestane in the osteoblasts may have been mediated by ametabolite of exemestane. Hence, a thorough investigationof exemestane and 17-hydroexemestane activities throughER and AR is warranted to provide evidence regardingwhether exemestane could display a more favorable safetyand toxicity profile than nonsteroidal AIs for long-termadjuvant use and as a chemopreventive of breast cancer inpostmenopausal women. Therefore, we evaluated thepharmacologic actions of exemestane and its primarymetabolite 17-hydroexemestane on ER- and AR-regulatedactivities in a range of cellular and molecular assays. First,we determined the relative binding affinity (RBA) of17-hydroexemestane to ERa and AR. Next, using MCF-7and T47D breast cancer cells, we examined the ability of17-hydroexemestane to stimulate cell proliferation and cellcycle progression (Supplementary Material)4 via ER andAR, to regulate ER- and AR-dependent transcription, andto modulate ERa and AR protein levels. Lastly, weinvestigated intermolecular interactions between 17-hydro-exemestane and ERa and AR using molecular modeling.

Materials andMethodsCompounds and Cell LinesExemestane and 17-hydroexemestane were provided by

Pfizer. Fulvestrant (ICI 182,780, Faslodex) and bicalutamide(Casodex) were provided by Dr. Alan E. Wakeling andDr. Barrington J.A. Furr (AstraZeneca Pharmaceuticals,Macclesfield, United Kingdom), respectively. All other

4 Supplementary material for this article is available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Androgenic and Estrogenic Effects of 17-HydroexemestaneOF2





compounds were obtained from Sigma-Aldrich, and cellculture reagents were from Invitrogen. All test agents weredissolved in ethanol and added to the medium at 1:1,000(v/v). MCF-7/WS8 and T47D:A18 human mammarycarcinoma cells, clonally selected from their parentalcounterparts for sensitivity to growth stimulation by E2(32), were used in all experiments indicating MCF-7 andT47D cells. Cells were maintained in steroid-replete RPMI1640, but 3 days before all experiments, were cultured insteroid-free media as previously described (32, 33).

Competitive Hormone-Binding AssaysCompetitive hormone-binding assays were conducted

using fluorescence polarization–based ERa and AR Com-petitor Assay kits (Invitrogen) as previously described (34).

Cellular Proliferation AssaysCellular proliferation following 7 days in culture was

determined by DNA mass per well in 12-well plates usingthe fluorescent DNA dye Hoechst 33258 as previouslydescribed (32).

Reporter GeneAssaysReporter gene assays were conducted by transfecting

cells with either an ERE(5x)-regulated (pERE(5x)TA-ffLuc;ref. 33) or ARE(5x)-regulated (pAR-Luc; Panomics) fireflyluciferase expression plasmid and co-transfected with abasal TATA promoter-regulated (pTA-srLuc) Renilla lucif-erase expression plasmid as previously described (33).

Quantitative Real-Time PCRQuantitative real-time PCR (qPCR) was used to deter-

mine AR and ribosomal large phosphoprotein subunit P0(RLP0; 36B4) mRNA levels as previously described (35).

Immunoblot AnalysesImmunoblots, prepared as previously described (33),

were probed with primary antibodies against AR (AR 441;Lab Vision), ERa (AER 611; Lab Vision), and h-actin(AC-15; Sigma-Aldrich).

Molecular Modeling andVirtual Docking CalculationsThe three-dimensional conformations for E2, 17-hydro-

exemestane, exemestane, R1881, and dexamethasone weregenerated with Omega version 2.1 software (OpenEyeScientific Software). These compounds were docked usingthe following X-ray crystallographic structures: 1GWR(ERa co-complexed with E2, 2.4-Å resolution; ref. 36) and1XQ3 (AR co-complexed with R1881, 2.25-Å resolution;ref. 37). ERa and AR ligand-binding pockets were builtusing a ligand-centered box and the receptor-boundconformation of the respective ligand: E2 (for 1GWR) andR1881 (for 1XQ3). The volume of the cavity differs for thetwo receptors: 648 Å3 for 1GWR and 532 Å3 for 1XQ3. Allreceptor and ligand bonds were kept rigid. The receptorstructures were filled with water because ERa (38) andAR crystal structures (39) indicate that specific stablehydrogen bond (H-bond) networks form among particularwater molecules, ligands, and amino acid side chains.Docking was done with FRED version 2.2 software (Open-Eye) using a short refinement step for the ligands withinthe receptor and using the MMFF94 force field. The best30 conformations for each compound were compared andranked by FRED’s Chemscore function. For each ligand-

docked receptor evaluated, the docked conformation withthe lowest total intermolecular interaction energy (kJ/mol)was selected. To address whether water could be displacedby a compound during the process of binding, dockingcalculations were also done using receptors modeled withwater removed as presented in Supplementary Table S14

and the differences between the methods in SupplementaryTable S2.4

Curve Fitting and Statistical AnalysesAll statistical tests, curve fitting, and determination of

half-maximal inhibitory concentrations (IC50) and half-maximal effective concentrations (EC50) were done usingGraphPad Prism 4.03 (GraphPad Software). Significantdifferences were determined using one-way ANOVA withBonferroni multiple comparison post-test.

ResultsExperimentally Determined Binding of 17-Hydro-

exemestane and Exemestane to ERA and ARStructures of the compounds relevant to these studies,

the steroidal AI parent compound exemestane, its primarymetabolite 17-hydroexemestane, E2, and the syntheticnon-aromatizable androgen R1881, are shown in Fig. 1A.Importantly, the only difference between parental exemes-tane and its metabolite 17-hydroexemestane is a hydroxylgroup in the metabolite in place of a ketone in the parentcompound at the 17h position, whereas both compoundsshare a 3-keto group. For steroidal estrogens, elimination ormodification of the 17h-OH group reduces binding to ERa,but that of the 3-OH group is much more dramatic (40).For steroidal androgens, the trend is reversed; eliminationor modification of the 17h-OH group is more significant forAR binding than that of the 3-keto group (41). The 3-ketogroup found in both exemestane and 17-hydroexemestanealso favors binding to AR (41).We tested the binding of exemestane and 17-hydroexe-

mestane to ERa and AR using fluorescence polarization–based competitive hormone-binding assays (Fig. 1B and C;Table 1). For purposes of comparison, compound affinitieswere arbitrarily categorized with respect to their RBAs asstrong (100 to z1), moderate (

17-hydroexemestane as a strong and exemestane as a weakAR ligand. However, dexamethasone would also becategorized as a weak AR ligand. Hence, the observedvery weak ERa binding and strong AR binding of 17-hydroexemestane was consistent with what previouslyreported structure-activity relationships (40, 41) wouldhave predicted due to reduction of the 17-keto group inexemestane to a 17h-OH in the metabolite.

Proliferation Responses to 17-Hydroexemestane andExemestaneWe examined the effects of exemestane and 17-hydro-

exemestane on 7 days of proliferation in ERa- andAR-positive MCF-7 and T47D mammary carcinoma cells(Fig. 2). As expected, both cell lines were growth stimulatedby E2, with growth EC50s of 1.7 � 10�12 mol/L E2 for MCF-7 cells (Fig. 2A) and 7.1 � 10�12 mol/L E2 for T47D cells(Fig. 2B). These growth responses to E2 were completelyblocked by fulvestrant (all P values

concentrations and via ER at higher concentrations. Theseresults were consistent with the observed binding affinitiesof these compounds to ERa (Fig. 1B) and AR (Fig. 1C).Cell Cycle Progression Responses to 17-Hydroexe-

mestaneAs shown in Supplementary Fig. S1,4 17-hydroexemes-

tane at 10�8 mol/L acted through AR to stimulate S-phaseentry in T47D cells by 1.9-fold (P < 0.001) but, at 5 � 10�6mol/L, acted through ER to stimulate S-phase entry inMCF-7 cells by 2.2-fold (P < 0.001). Hence, 17-hydro-exemestane effects on cell cycle progression were consistentwith its effects on proliferation (Fig. 2).

Regulation of ERA and AR Transcriptional Activitiesby17-HydroexemestaneNext, we investigated the ability of 17-hydroexemestane

to regulate ER and AR transcriptional activity by trans-fecting cells with an ERE(5x)-regulated or ARE(5x)-regulated dual-luciferase plasmid set, treating cells withtest compounds, and measuring dual-luciferase activity44 h after treatment (Fig. 3A–C). E2 at 10

�10 mol/L inducedERE(5x)-regulated transcription by 19.4-fold in MCF-7 cells(Fig. 3A; P < 0.001), and 11.3-fold in T47D cells (Fig. 3B;P < 0.001) compared with control-treated cells; this E2-induced transcriptional activity was blocked by fulvestrant(both P values

AR mRNA expression by 48% (P < 0.001), whereas10�9 mol/L E2 did not (Fig. 3D). Bicalutamide preventedR1881-mediated decrease inARmRNAexpression (Fig. 3D),validating that AR mRNA levels were negatively feedbackregulated. Similarly, a low 10�8 mol/L concentration of17-hydroexemestane led to a 41% decrease in AR mRNAlevels (P < 0.01), with increased 17-hydroexemestaneconcentrations further decreasing AR mRNA expression(Fig. 3D). Bicalutamide blocked 17-hydroexemestane–mediated down-regulation of AR mRNA expression(P < 0.01), whereas fulvestrant did not (Fig. 3D). Therefore,17-hydroexemestane acted as an androgen via AR tofeedback-regulate the expression of endogenous AR mRNAin T47D cells.

Modulation of AR and ERA Protein Levels by17-HydroexemestaneAndrogens and estrogens modulate protein expression

levels of their cognate receptors. R1881 stabilizes ARprotein allowing its accumulation (43), whereas E2 pro-motes ERa degradation in a cell type–dependent manner(32). Therefore, we investigated the effects of 17-hydro-exemestane on AR and ERa protein levels by treating cellswith test compounds for 24 h and analyzing receptor levelsby immunoblotting. E2 decreased ERa protein levels inMCF-7 (Fig. 4A), but not T47D cells (Fig. 4B), as we havepreviously shown (32). As expected, fulvestrant promotedERa protein degradation in both cell lines. E2 did notsignificantly affect AR protein accumulation in MCF-7 cells

Figure 2. 17-Hydroexemestane and R1881 stimulate cellular proliferation. DNA-based cellular proliferation assays of (A) MCF-7 cells treated with E2and R1881, (B) T47D cells treated with E2 and R1881, (C) MCF-7 cells treated with exemestane and 17-hydroexemestane, and (D) T47D cells treated withexemestane and 17-hydroexemestane. Cells were cultured in steroid-free medium for 3 d before the assays. MCF-7 cells were seeded at 15,000 cells perwell and T47D cells at 20,000 cells per well in 12-well plates. Cells were treated on days 0 (the day after seeding), 3, and 6, and then collected on day 7.Cellular DNA quantities were determined using the fluorescent DNA-binding dye Hoechst 33258 and compared against a standard curve. Data shownrepresent the mean of four replicates and SDs. DNA values were fitted to a sigmoidal dose-response curve and growth EC50s calculated using GraphPadPrism 4.03 software. At high concentrations, 17-hydroexemestane and R1881 increased growth via ER in both cell lines but, at low concentrations,stimulated growth via AR selectively in T47D cells. Abbreviations: CON, control; FUL, fulvetsrant; BIC, bicalutamide.






(Fig. 4A), but did down-regulate AR protein levels in T47Dcells (Fig. 4B). Also, fulvestrant and E2 plus fulvestranttreatments did not significantly affect AR protein levelsin MCF-7 cells (Fig. 4A), but did modestly up-regulate ARprotein levels in T47D cells (Fig. 4B). As expected, R1881caused an increase in accumulation of AR protein in bothcell lines (Fig. 4A and B), likely by stabilizing the protein(43). Next, we characterized the effects of low 10�8 mol/Land high 5 � 10�6 mol/L concentrations of 17-hydro-exemestane on ERa and AR expression. The high 5 � 10�6mol/L concentration of 17-hydroexemestane led to de-creased ERa protein levels in MCF-7 (Fig. 4A), but not inT47D cells (Fig. 4B); this pattern indicates that 5� 10�6mol/L

17-hydroexemestane acted as an estrogen to regulate ERaprotein in a cell type–dependent manner. Similar to R1881,treatment with low 10�8 mol/L or high 5 � 10�6 mol/Lconcentrations of 17-hydroexemestane led to increased ARprotein accumulation in both cell lines (Fig. 4A and B),indicating that 17-hydroexemestane acted as an androgenlikely by stabilizing AR protein. Therefore, 17-hydroexemes-tane modulated ERa and AR protein accumulation as wouldan estrogen and an androgen, respectively.

Molecular Docking of 17-Hydroexemestane andExemestane to ERA and ARTo investigate the mechanism by which 17-hydroexe-

mestane binds ERa as a very weak ligand and AR as a

Figure 3. 17-Hydroexemestane and R1881 regulate ER transcriptional activity at high concentrations and AR transcriptional activity at lowconcentrations. ERE(5x)-regulated dual-luciferase activity in (A) MCF-7 cells and (B) T47D cells. (C) ARE(5x)-regulated reporter gene activity in T47D cells.A–C , Under steroid-free conditions, cells were transiently transfected with pERE(5x)TA-ffLuc or pARE(5x)-Luc (firefly luciferase reporter plasmids) and theinternal normalization control pTA-srLuc (Renilla luciferase reporter plasmid). Four hours after transfection, cells were treated as indicated and then againthe following day. Cells were assayed 44 h after transfection for dual-luciferase activity. Data shown are the mean of triplicate determinations andassociated SDs. 17-Hydroexemestane and R881 stimulated ERE(5x)-regulated transcription in MCF-7 and T47D cells and ARE(5x)-regulated transcriptionalactivity in T47D cells. D, AR mRNA levels in T47D cells as determined by real-time PCR. T47D cells were treated as indicated for 24 h. RNA was isolatedand converted to cDNA. Continuous accumulation of PCR products was monitored using the double strand-specific DNA dye SYBR Green. Quantitativemeasurements of AR mRNA and the endogenous normalization control RLP0 mRNA were determined by comparison to a standard curve of knownquantities of serially diluted AR or RLP0 PCR product. The data represent the mean and SDs of three independent samples, each of which was measured intriplicate. 17-Hydroexemestane and R881 down-regulated AR mRNA levels at nanomolar concentrations in an AR-dependent manner.

Molecular Cancer Therapeutics OF7





strong ligand, molecular models were constructed in silico .The trends in the computed intermolecular interactionenergies matched the experimentally determined RBAs(Table 1). Superimposition of the docked and crystallo-graphic structures of E2 complexed with ERa (Fig. 5A) andof R1881 complexed with AR (Fig. 5B) showed that thedocking models recapitulated the molecular recognitionpatterns of the crystal structures.Considering ERa, the intermolecular interaction energies

of R1881 and 17-hydroexemestane were less favorable thanE2 by 1.94 and 2.76 kJ/mol, respectively, due to decreasedH-bond interactions and increased steric clash (Table 1).Exemestane was much less favorable than E2 by 4.57 kJ/mol(Table 1). Hence, the 17h-OH group of 17-hydroexemestanecompared with the 17-keto group of exemestane contri-buted �1.81 kJ/mol toward increased affinity for ERa.Interestingly, the docking calculations suggested that thehigher affinity of 17-hydroexemestane over exemestane forERa was not due to increased H-bonding mediated by the17h-OH group, but rather increased lipophilic interactions(Table 1) due to a slight repositioning of the compoundas a consequence of 17h-OH group. In the E2 docked to ERamodel, H-bonds between E2 and Glu

353, Arg394, and His524

side chains were observed (Fig. 5A). In the docked 17-hydroexemestane to ERa model (Fig. 5C), the same Arg394

and His524 interactions were maintained, except that therewas a loss of the Glu353 interaction. The R1881 docked toERa model is shown in Supplementary Fig. S2A.4

Considering AR, the intermolecular interaction energy of17-hydroexemestane was only 0.8 kJ/mol less favorable

than R1881, whereas exemestane was significantly lessfavorable than R1881 by 6.27 kJ/mol (Table 1). Docking of17-hydroexemestane to AR, compared with the parent drugexemestane, indicated that 17-hydroexemestane exhibitedimproved lipophilic interactions by �2.11 kJ/mol, morefavorable H-bonding interactions by �2.65 kJ/mol, anddecreased steric clash by �1.08 kJ/mol. Hence, the 17h-OHgroup in 17-hydroexemestane compared with the 17-ketogroup in exemestane contributed �5.47 kJ/mol towardhigher affinity for binding AR (Table 1). In the R1881docked to AR model, H-bonds between R1881 and Asn705,Gln711 and Arg752 were observed (Fig. 5B). The OH sidechain of Thr877 was in close proximity to both docked R1881(Fig. 5B) and 17-hydroexemestane (Fig. 5D), but the anglewas not favorable for H-bonding. Docking of 17-hydro-exemestane to AR (Fig. 5D) indicated a short 2.78-ÅH-bond between the 17h-OH group of the ligand andAsn705, but not between the 3-keto group of the ligandand Gln711 and Arg752. Hence, the short 2.78-Å H-bondobserved in the 17-hydroexemestane docked to ARmodel was important in mediating high affinity binding.The exemestane docked to AR model is shown inSupplementary Fig. S2B.4

DiscussionWe observed that 17-hydroexemestane, the primary metab-olite of exemestane, bound to ERa as a very weakligand and acted through ER at high sub-micromolar andmicromolar concentrations to stimulate growth, promotecell cycle progression, induce ERE-regulated reporter geneexpression, and down-modulate ERa protein levels inbreast cancer cells. However, we also observed that 17-hydroexemestane bound to AR as a strong ligand and foundin T47D cells that 17-hydroexemestane stimulated growth,induced cell cycle progression, down-modulated ARmRNAexpression, and stabilized AR protein levels, with all ofthese effects occurring at low nanomolar concentrationsand blocked by bicalutamide. Moreover, computer dockingindicated that the 17h-OH group of 17-hydroexemestaneversus the 17-keto group of exemestane contributedsignificantly more toward increasing affinity to AR than toERa. Molecular modeling also indicated that 17h-OH groupof 17-hydroexemestane interacted with AR through animportant H-bond of Asn705, a conserved recognition motifemployed by R1881. Therefore, we propose that the primarymechanism of action of exemestane in vivo is mediated by17-hydroexemestane regulating AR activities.The Food and Drug Administration label for exemestane

(Aromasin; Pfizer) reports that in postmenopausal womenwith advanced breast cancer, the mean AUC (area underthe curve) values of exemestane following repeated doseswas 75.4 ng�h/mL (254 nmol�h/L), which was almost twicethat in healthy postmenopausal women (41.4 ng�h/mL;140 nmol�h/L; ref. 31). Because circulating levels of 17-hydroexemestane can reach about 1/10 the level of theparent compound (30), we hypothesize that circulatinglevels of 17-hydroexemestane are sufficient to bind AR and

Figure 4. 17-Hydroexemestane modulates AR and ERa protein levels.Immunoblot analysis of AR and ERa in (A) MCF-7 cells and (B) T47D cells.Cells were treated as indicated for 24 h, and 20 Ag of cellular protein wereresolved by 4% to 12% SDS-PAGE and then transferred to a nylonmembrane. Membranes were probed for AR, ERa, and h-actin, andimmunoreactive bands were visualized by chemiluminescence andautoradiography. Cropped blots are shown. 17-hydroexemestane up-regulated AR protein levels at 10�8 mol/L in both cell lines and down-regulated ERa in MCF-7 cells at 5 � 10�6 mol/L.






regulate AR-dependent activities. Furthermore, a subpop-ulation of patients may exist who metabolize exemestaneat higher rates, leading to correspondingly higher circulat-ing 17-hydroexemestane levels. For instance, one of threepatients administered 800 mg of exemestane, the highestdose evaluated, achieved 17-hydroexemestane plasmalevels approximately one-half the level of the parentcompound (30). Based on our results, we would predictthat higher circulating levels of 17-hydroexemestane wouldassociate with decreased rates of BMD loss and risk of bonefractures in postmenopausal women. We suggest thatcirculating levels of 17-hydroexemestane and exemestaneshould be determined in clinical trials and correlated todisease outcome and toxicity profiles such as BMD loss.Although the clinical studies reported thus far were not

designed to directly compare one AI versus another, com-parisons in the rate of BMD loss from baseline to year 1,and from year 1 to 2 can be made. In the bone safetysubprotocol of the IES (Intergroup Exemestane Study) trial,

the rate of BMD loss was greatest within 6 months ofswitching from tamoxifen to exemestane at �2.7% in thelumbar spine and �1.4% in the hip, but thereafter, BMDloss progressively slowed in months 6 to 12 and again inmonths 12 to 24 to only �1.0% and �0.8% in the lumbarspine and hip, respectively (10), which is in the same rangeas would be expected for postmenopausal women ingeneral. However, in the bone safety substudy of theMA.17 trial, patients administered letrozole experienceda relatively constant rate of BMD loss for 2 years: at12 months, the rate of BMD loss from baseline was �3.3%and �1.43% in lumbar spine and hip, respectively, andfrom year 1 to year 2, �2.05% and �2.17% in lumbar spineand hip, respectively (11). In the bone substudy of theATAC (Arimidex, Tamoxifen, Alone or in Combination)trial, the rate of BMD loss from baseline to year 1 was�2.2% in lumbar spine and �1.5% in hip and from year 1 toyear 2, �1.8% in lumbar spine and �1.9% in hip (18).Collectively, these results suggest that after the initial

Figure 5. Intermolecular interactions of ligands complexed with ERa and AR by computer docking. A, superposition of E2 from the X-ray crystalstructure (gray ) and modeled E2 (yellow ) docked to ERa. B, superposition of R1881 from the crystal structure (gray ) and modeled R1881 (yellow ) dockedto AR. C, modeled 17-hydroexemestane docked to ERa. D, modeled 17-hydroexemestane docked to AR. Cyan, red, and blue, hydrogen, oxygen, andnitrogen atoms, respectively. Green, carbon backbone of the protein. Hydrogens from the X-ray crystal conformations of E2 (A) and R1881 (C) wereomitted. H-bonds were shown to the modeled compound conformations only. Dashed lines, intermolecular H-bonds up to 3.5 Å; their length in angstromsis indicated.






12months of AI therapy, exemestanemay be associatedwithslower rates of BMD loss compared with nonsteroidal AIs.Furthermore, although not directly comparable, the fracturerate per 1,000 woman-years in the ATAC trial was 22.6 foranastrozole and 15.6 for tamoxifen (1), whereas in the IEStrial, the incidence rate per 1,000 woman-years for multiplefractures was 19.2 for exemestane and 15.1 for tamoxifen(10). These results show that although both anastrozoleand exemestane were associated with higher fracture ratesthan tamoxifen, they also suggest that exemestane may beassociated with a lower fracture rate than anastrozole.Clinical trials now under way to directly compare thedifferent AIs will hopefully provide clear results.Androgens regulate growth of normal and neoplastic

mammary cells in a cell type-specific manner, either byinhibiting or stimulating growth (44). However, themechanisms by which androgens via AR regulate breastcancer growth remain elusive. Female AR knock-out miceexhibit decreased ductal branching and terminal end budsin prepubertal animals and retarded lobuloalveolar devel-opment in adult animals (45). Likewise, targeted disruptionof AR in MCF-7 cells also leads to severe inhibition ofproliferation (45). Epidemiologic analyses indicate a posi-tive correlation between androgen levels and the incidenceof breast cancer; meta-analysis from nine prospectivestudies showed that a doubling in testosterone concen-trations in postmenopausal women translated into anincreased relative risk of 1.42 unadjusted and 1.32 adjustedfor E2 (46). AR status in breast cancer associates with bothpositive and negative indicators and clinical outcome. ARexpression has been found in 84% (47) to 91% (48) ofclinical breast cancers, and associated with ER status, buthas also been found in 49% of ER-negative tumors (49).Patients with tumors that coexpress AR with ER andprogesterone receptor have shown longer disease-freesurvival (DFS) than patients whose tumors were negativefor all three receptors (48), but AR protein levels have alsoserved as an independent predictor of axillary metastasesin multivariate analysis (47) Furthermore, AR expressionhas correlated with decreased histopathologic grade,greater age, and postmenopausal status, but also lymphnode–positive status (50). In AR-positive/ER-negativetumors, AR expression again associated with positive andnegative indicators/outcome such as increased age, post-menopausal status, and longer DFS but also tumor grade,tumor size, and HER-2/neu overexpression (49).Patients who fail AI therapy, whether the AI was

steroidal or nonsteroidal, likely harbor tumor cells thathave been selected for growth in an estrogen-depletedenvironment and, hence, are not dependent on ER activityfor survival. Not all androgens are metabolized byaromatase to estrogens; for instance, dihydrotestosteronecannot be converted to an estrogen by aromatase (44).Thus, a possible mechanism for failure of AI therapy in theclinic is androgen-stimulated breast cancer growth, alargely unrecognized alternative mechanism. We observedcellular proliferation of T47D cells in response to R1881 and17-hydroexemestane, and these effects were blocked by

bicalutamide. Therefore, T47D cells contain a functional ARsignaling pathway that promoted growth in the absence ofestrogen. Because functional AR signaling could beetiologically involved in a subpopulation of clinical breastcancers, those patients who have AR-positive tumors andachieve high circulating levels of 17-hydroexemestane, yetwhose disease progresses while on exemestane therapy,may respond to AR-based therapy such as the antiandro-gen bicalutamide.

Acknowledgments

We thank Dr. Alan E. Wakeling and Dr. Barrington J.A. Furr for providingfulvestrant and bicalutamide, respectively. We also thank members ofthe Jordan laboratory for helpful discussions, and Dr. Jennifer L. Ariazi(GlaxoSmithKline, Collegeville, PA) for critical review of the manuscript.

References

1. Howell A, Cuzick J, Baum M, et al. Results of the ATAC (Arimidex,Tamoxifen, Alone or in Combination) trial after completion of 5 years’adjuvant treatment for breast cancer. Lancet 2005;365:60–2.

2. Jonat W, Gnant M, Boccardo F, et al. Effectiveness of switching fromadjuvant tamoxifen to anastrozole in postmenopausal women withhormone-sensitive early-stage breast cancer: a meta-analysis. LancetOncol 2006;7:991–6.

3. Coates AS, Keshaviah A, Thurlimann B, et al. Five years of letrozolecompared with tamoxifen as initial adjuvant therapy for postmenopausalwomen with endocrine-responsive early breast cancer: update of studyBIG 1–98. J Clin Oncol 2007;25:486–92.

4. Goss PE, Ingle JN, Martino S, et al. Randomized trial of letrozolefollowing tamoxifen as extended adjuvant therapy in receptor-positivebreast cancer: updated findings from NCIC CTG MA.17. J Natl Cancer Inst2005;97:1262–71.

5. Coombes RC, Hall E, Gibson LJ, et al. A randomized trial of exemestaneafter two to three years of tamoxifen therapy in postmenopausal womenwith primary breast cancer. N Engl J Med 2004;350:1081–92.

6. Coombes RC, Kilburn LS, Snowdon CF, et al. Survival and safety ofexemestane versus tamoxifen after 2–3 years’ tamoxifen treatment(Intergroup Exemestane Study): a randomised controlled trial. Lancet2007;369:559–70.

7. Jordan VC, Brodie AM. Development and evolution of therapiestargeted to the estrogen receptor for the treatment and prevention ofbreast cancer. Steroids 2007;72:7–25.

8. Howell A, Clarke RB, Evans G, et al. Estrogen deprivation for breastcancer prevention. Recent Results Cancer Res 2007;174:151–67.

9. Buzdar A, Howell A, Cuzick J, et al. Comprehensive side-effect profileof anastrozole and tamoxifen as adjuvant treatment for early-stage breastcancer: long-term safety analysis of the ATAC trial. Lancet Oncol 2006;7:633–43.

10. Coleman RE, Banks LM, Girgis SI, et al. Skeletal effects ofexemestane on bone-mineral density, bone biomarkers, and fractureincidence in postmenopausal women with early breast cancer participatingin the Intergroup Exemestane Study (IES): a randomised controlled study.Lancet Oncol 2007;8:119–27.

11. Perez EA, Josse RG, Pritchard KI, et al. Effect of letrozole versusplacebo on bone mineral density in women with primary breast cancercompleting 5 or more years of adjuvant tamoxifen: a companion study toNCIC CTG MA.17. J Clin Oncol 2006;24:3629–35.

12. Eastell R, Hannon RA, Cuzick J, Dowsett M, Clack G, Adams JE.Effect of an aromatase inhibitor on bmd and bone turnover markers: 2-yearresults of the Anastrozole, Tamoxifen, Alone or in Combination (ATAC)trial (18233230). J Bone Miner Res 2006;21:1215–23.

13. Morales L, Pans S, Paridaens R, et al. Debilitating musculoskeletalpain and stiffness with letrozole and exemestane: associated tenosynovialchanges on magnetic resonance imaging. Breast Cancer Res Treat2007;104:87–91.

14. Mackey J, Gelmon K. Adjuvant aromatase inhibitors in breast cancertherapy: significance of musculoskeletal complications. Curr Opin Oncol2007;19 Suppl 1:S9–18.






15. Goss PE, Qi S, Josse RG, et al. The steroidal aromatase inhibitorexemestane prevents bone loss in ovariectomized rats. Bone 2004;34:384–92.

16. Goss PE, Qi S, Cheung AM, Hu H, Mendes M, Pritzker KP. Effects ofthe steroidal aromatase inhibitor exemestane and the nonsteroidalaromatase inhibitor letrozole on bone and lipid metabolism in ovariecto-mized rats. Clin Cancer Res 2004;10:5717–23.

17. Lonning PE, Geisler J, Krag LE, et al. Effects of exemestaneadministered for 2 years versus placebo on bone mineral density, bonebiomarkers, and plasma lipids in patients with surgically resected earlybreast cancer. J Clin Oncol 2005;23:5126–37.

18. Coleman RE, Group AT. Effect of anastrozole on bone mineraldensity: 5-year results from the ‘Arimidex,’ Tamoxifen, Alone or inCombination (ATAC) trial. J Clin Oncol (Meeting Abstracts) 2006;24:511.

19. Love RR, Mazess RB, Barden HS, et al. Effects of tamoxifen on bonemineral density in postmenopausal women with breast cancer. N Engl JMed 1992;326:852–6.

20. Cummings SR, Browner WS, Bauer D, et al. Endogenous hormonesand the risk of hip and vertebral fractures among older women. N Engl JMed 1998;339:733–8.

21. Hofbauer LC, Khosla S. Androgen effects on bone metabolism: recentprogress and controversies. Eur J Endocrinol 1999;140:271–86.

22. Miki Y, Suzuki T, Hatori M, et al. Effects of aromatase inhibitors onhuman osteoblast and osteoblast-like cells: a possible androgenic boneprotective effects induced by exemestane. Bone 2007;40:876–87.

23. Lea CK, Flanagan AM. Physiological plasma levels of androgensreduce bone loss in the ovariectomized rat. Am J Physiol 1998;274:E328–35.

24. Tobias JH, Gallagher A, Chambers TJ. 5 a-Dihydrotestosteronepartially restores cancellous bone volume in osteopenic ovariectomizedrats. Am J Physiol 1994;267:E853–9.

25. Buchanan JR, Hospodar P, Myers C, Leuenberger P, Demers LM.Effect of excess endogenous androgens on bone density in young women.J Clin Endocrinol Metab 1988;67:937–43.

26. Dixon JE, Rodin A, Murby B, Chapman MG, Fogelman I. Bone mass inhirsute women with androgen excess. Clin Endocrinol (Oxf) 1989;30:271–7.

27. Riggs BL, Jowsey J, Goldsmith RS, Kelly PJ, Hoffman DL, Arnaud CD.Short- and long-term effects of estrogen and synthetic anabolic hormonein postmenopausal osteoporosis. J Clin Invest 1972;51:1659–63.

28. Barrett-Connor E, Young R, Notelovitz M, et al. A two-year, double-blind comparison of estrogen-androgen and conjugated estrogens insurgically menopausal women. Effects on bone mineral density, symptomsand lipid profiles. J Reprod Med 1999;44:1012–20.

29. di Salle E, Giudici D, Ornati G, et al. 4-Aminoandrostenedionederivatives: a novel class of irreversible aromatase inhibitors. Comparisonwith FCE 24304 and 4-hydroxyandrostenedione. J Steroid Biochem MolBiol 1990;37:369–74.

30. Evans TR, Di Salle E, Ornati G, et al. Phase I and endocrine study ofexemestane (FCE 24304), a new aromatase inhibitor, in postmenopausalwomen. Cancer Res 1992;52:5933–9.

31. Pfizer Inc. Product information: Aromasin, exemestane tablets.New York, NY 10017; 2007.

32. Pink JJ, Jordan VC. Models of estrogen receptor regulation byestrogens and antiestrogens in breast cancer cell lines. Cancer Res 1996;56:2321–30.

33. Ariazi EA, Kraus RJ, Farrell ML, Jordan VC, Mertz JE. Estrogen-related receptor a1 transcriptional activities are regulated in part via theErbB2/HER2 signaling pathway. Mol Cancer Res 2007;5:71–85.

34. Abdelrahim M, Ariazi E, Kim K, et al. 3-Methylcholanthrene and otheraryl hydrocarbon receptor agonists directly activate estrogen receptor a.Cancer Res 2006;66:2459–67.

35. Bauer JA, Thompson TA, Church DR, Ariazi EA, Wilding G. Growthinhibition and differentiation in human prostate carcinoma cells induced bythe vitamin D analog 1a,24-dihydroxyvitamin D2. Prostate 2003;55:159–67.

36. Warnmark A, Treuter E, Gustafsson JA, Hubbard RE, Brzozowski AM,Pike AC. Interaction of transcriptional intermediary factor 2 nuclearreceptor box peptides with the coactivator binding site of estrogenreceptor a. J Biol Chem 2002;277:21862–8.

37. He B, Gampe RT, Jr., Kole AJ, et al. Structural basis for androgenreceptor interdomain and coactivator interactions suggests a transitionin nuclear receptor activation function dominance. Mol Cell 2004;16:425–38.

38. Brzozowski AM, Pike AC, Dauter Z, et al. Molecular basis of agonismand antagonism in the oestrogen receptor. Nature 1997;389:753–8.

39. Matias PM, Donner P, Coelho R, et al. Structural evidence for ligandspecificity in the binding domain of the human androgen receptor.Implications for pathogenic gene mutations. J Biol Chem 2000;275:26164–71.

40. Fang H, Tong W, Shi LM, et al. Structure-activity relationships for alarge diverse set of natural, synthetic, and environmental estrogens. ChemRes Toxicol 2001;14:280–94.

41. Fang H, Tong W, Branham WS, et al. Study of 202 natural, synthetic,and environmental chemicals for binding to the androgen receptor. ChemRes Toxicol 2003;16:1338–58.

42. Wolf DA, Herzinger T, Hermeking H, Blaschke D, Horz W. Transcrip-tional and posttranscriptional regulation of human androgen receptorexpression by androgen. Mol Endocrinol 1993;7:924–36.

43. Zhou ZX, Lane MV, Kemppainen JA, French FS, Wilson EM.Specificity of ligand-dependent androgen receptor stabilization: receptordomain interactions influence ligand dissociation and receptor stability.Mol Endocrinol 1995;9:208–18.

44. Somboonporn W, Davis SR. Testosterone effects on the breast:implications for testosterone therapy for women. Endocr Rev 2004;25:374–88.

45. Yeh S, Hu Y-C, Wang P-H, et al. Abnormal mammary glanddevelopment and growth retardation in female mice and MCF7 breastcancer cells lacking androgen receptor. J Exp Med 2003;198:1899–908.

46. Group EHaBCC. Endogenous sex hormones and breast cancer inpostmenopausal women: reanalysis of nine prospective studies. J NatlCancer Inst 2002;94:606–16.

47. Soreide JA, Lea OA, Varhaug JE, Skarstein A, Kvinnsland S.Androgen receptors in operable breast cancer: relation to other steroidhormone receptors, correlations to prognostic factors and predictive valuefor effect of adjuvant tamoxifen treatment. Eur J Surg Oncol 1992;18:112–8.

48. Kuenen-Boumeester V, Van der Kwast TH, Claassen CC, et al. Theclinical significance of androgen receptors in breast cancer and theirrelation to histological and cell biological parameters. Eur J Cancer 1996;32A:1560–5.

49. Agoff SN, Swanson PE, Linden H, Hawes SE, Lawton TJ. Androgenreceptor expression in estrogen receptor-negative breast cancer. Immu-nohistochemical, clinical, and prognostic associations. Am J Clin Pathol2003;120:725–31.

50. Bieche I, Parfait B, Tozlu S, Lidereau R, Vidaud M. Quantitation ofandrogen receptor gene expression in sporadic breast tumors by real-timeRT-PCR: evidence that MYC is an AR-regulated gene. Carcinogenesis2001;22:1521–6.






Published OnlineFirst November 7, 2007.Mol Cancer Ther effects as an androgenExemestane's 17-hydroxylated metabolite exerts biological

Updated version

10.1158/1535-7163.MCT-07-0312doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2007/12/14/1535-7163.MCT-07-0312.DC1

Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://mct.aacrjournals.org/content/early/2005/11/01/1535-7163.MCT-07-0312.citationTo request permission to re-use all or part of this article, use this link


http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-07-0312http://mct.aacrjournals.org/content/suppl/2007/12/14/1535-7163.MCT-07-0312.DC1http://mct.aacrjournals.org/cgi/alertsmailto:[email protected]://mct.aacrjournals.org/content/early/2005/11/01/1535-7163.MCT-07-0312.citationhttp://mct.aacrjournals.org/

exemestane’s17-hydroxylatedmetaboliteexertsbiological ... › content › molcanther › ...nov...

Documents